Project Summary/Abstract Arsenic is a highly toxic environmental contaminant that poses a potential health threat to millions of people worldwide. Prenatal exposure to high levels of arsenic has been linked to multiple adverse infant health outcomes, including spontaneous abortion, stillbirth, reduced birth weight and increased rates of infection. Moreover, growing evidence suggests that even levels of arsenic close to the Environmental Protection Agency's maximum contaminant level (EPA MCL) of 10 ?g/l, which are common in water supplies worldwide, can adversely affect infant health. Our research goal is to determine the effects of prenatal exposure to common levels of arsenic on fetal development, and elucidate the molecular mechanisms underlying these effects. We have begun to address these questions using data from the New Hampshire Birth Cohort Study, a study of pregnant women and their infants in a US region with elevated arsenic. Using samples from the fetal portion of the placenta, we performed gene expression analysis by RNA sequencing (RNA-seq), and identified several genes whose expression associates with arsenic exposure. Bioinformatic analysis related these effects to dysregulation of molecular and functional processes, consistent with this exposure resulting in adverse infant health outcomes. In the proposed project, we will use human embryonic stem cells (hESCs) as an experimental model to further investigate the effects of arsenic exposure on the developing human fetus. In Aim 1, we will identify the primary signaling nodes targeted by arsenic. We will use RNA-seq to analyze differential gene expression in hESCs after acute exposure to doses of arsenic at or below the EPA MCL. Using bioinformatic analysis of these data, we will identify arsenic-responsive signaling nodes, prioritize them based on overlaps with our data from fetal placenta, and validate arsenic's effects on these nodes in hESCs using qPCR. In Aim 2, we will determine the developmental effects of modulating the pivotal signaling nodes targeted by arsenic, identified in fetal placenta, or in hESCs in Aim 1. We will manipulate the expression of key components of these nodes in hESCs, by overexpression, knockdown, and knockout. These hESCs will then be used to generate embryoid bodies, as a model of fetal development. We will determine the effects of manipulating these signaling nodes on embryoid body proliferation and survival, and on their differentiation into various tissue lineages. Completion of these Aims will provide an integrated insight into the molecular pathways through which arsenic affects fetal development and health.